Cells are constantly receiving, integrating, and reacting to physical and molecular signals. These signals define the cellular microenvironment, which varies from location to location within in vivo systems and in vitro cultures. Spatiotemporial patterns of soluble factors influence cellular behaviors such as growth, migration, and differentiation. Mimicking these chemical and molecular patterns in vitro is of particular interest for those seeking to understand cellular regulation in vitro and in vivo. Current technologies allowing patterning of soluble factors in gradients and other concentration profiles are limited to microfluidic devices or culture in or under a hydrogel. However many standard cell culture procedures and protocols calling for patterning of surface associated factors require open systems.
In addition to heterogeneity in their microenvironments, within a population, cells exhibit individual-to-individual heterogeneity. As a result, measures of behaviors averaged over the population may not accurately reflect the state of a specific cell within that population. The differences have led to the emergence of single cell analysis tools. However, current procedures and readouts for these analyses are not ideal, as they either fail to provide the user historical information about a cells origin in terms of microenvironment or they limit throughput and analyte detection. For example, in flow cytometry, the expression of proteins may be measured on a cell-by-cell basis. But, any information regarding the location of where the cells where first removed from their microenvironment and any information about the cell's position within a culture are lost, thereby leaving analyses ignorant of the effects of diverse microenvironments on diverse cell populations.
Conversely to methods requiring the removal of cells from their microenvironment, image based analysis can be used to track cellular histories. These methods are limited in throughput, requiring continuous monitoring of a limited number of locations in cultures, and the microscope's filters and light source dictate the number of analytes that can be imaged. Thus, important measures often cannot be obtained simultaneously and those that can be obtained are evaluated using image analyses methods that are relatively computationally intensive.
Therefore, it is a primary object and feature of the present invention to provide a stamp and a method for transferring particles to cells of a cell culture.
It is a further object and feature of the present invention to provide a stamp and a method for transferring particles to cells of a cell culture that enables direct correlation between differences within a heterogeneous cell population and the position of given cells within a microenvironment.
It is a still further object and feature of the present invention to provide a stamp and a method for transferring particles to cells of a cell culture that is simple to use and inexpensive to manufacture.
In accordance with the present invention, a stamp is provided for transferring particles to cells of a cell culture. The stamp includes a body having upper and lower surfaces. The lower surface includes a recessed portion. A gel extends along the recessed portion of the lower surface of the body and includes the particles pre-patterned therein. The body is configured to move between a first position wherein the gel is isolated from the cells of the cell culture and a second position wherein the particles pre-patterned in the gel communicate with the cells of the cell culture.
The body includes first and second passages extending between the upper surface and the recessed portion of lower surface thereof. The gel is received in the first and second passages through the body. The gel in the first passage defines a first well. The first well is adapted for receiving a first solution therein. The gel in the second passage defines a second well. The second well is adapted for receiving a second solution therein. At least one of the first and second solutions includes the particles to be transferred.
In accordance with a further aspect of the present invention, a method of transmitting particles to cells of a cell culture is provided. The particles are patterned in a gel extending along a portion of a lower surface of a stamp. The stamp is positioned such that the particles in the gel extending along the lower surface of the stamp communicate with the cells of the cell culture. The particles in the gel are allowed to diffuse into the cells of the cell culture.
The stamp includes a body has an upper surface, a lower surface having a recessed portion, and first and second passages extending between the upper surface and the recessed portion of lower surface thereof. The gel extends along the recessed portion of the lower surface of the body. The body may also include first and second passages extending between the upper surface and the recessed portion of lower surface thereof. The gel is received in the first and second passages through the body. The gel in the first passage defines a first well. The first well is adapted for receiving a first solution therein. The gel in the second passage defines a second well. The second well is adapted for receiving a second solution therein.
The step of patterning the particles includes the additional step of depositing a first solution in the first well. The first solution includes the particles. Thereafter, the particles are allowed to diffuse into the gel. The particles in the gel form a gradient between the first and second passages in the body along the recessed portion of the lower surface. It is contemplated to deposit cell culture media on the cell culture prior to the step of positioning the stamp.
In accordance with a still further aspect of the present invention, a method of transmitting particles to cells of a cell culture is provided. The method includes the steps of forming a cell culture on a base and patterning the particles in a gel extending along a lower surface of a stamp. Cell culture media is deposited on the cell culture and the stamp is positioned in communication with the cell culture media such that the particles in gel extending along the lower surface of the stamp communicate with the cells of the cell culture. The particles in the gel are allowed to diffuse into the cells of the cell culture through the cell culture media.
The stamp includes a body has an upper surface, a lower surface having a recessed portion, and first and second passages extending between the upper surface and the recessed portion of lower surface thereof. The gel extends along the recessed portion of the lower surface of the body. The body also includes first and second passages extending between the upper surface and the recessed portion of lower surface thereof. The gel is also received in the first and second passages through the body. The gel in the first passage define a first well. The first well is adapted for receiving a first solution therein. The gel in the second passage defines a second well. The second well is adapted for receiving a second solution therein.
The step of patterning the particles includes the additional step of depositing a first solution in the first well. The first solution include the particles. The particles are allowed to diffuse into the gel and form a gradient between the first and second passages in the body along the recessed portion of the lower surface. The particles are first particles and the method may include the additional step of depositing a second solution in the second well. The second solution includes second particles. The second particles are allowed to diffuse into the gel. The second particles in the gel form a second gradient from the between the second well and the first well in the body along the recessed portion of the lower surface.